U.S. patent number 11,419,841 [Application Number 16/762,739] was granted by the patent office on 2022-08-23 for uses of lipophenolic compounds.
This patent grant is currently assigned to Centre National de la Recherche Scientifique (CNRS), Ecole Nationale Superieure de Chimie, Institut de Recherche Pour le Developpement (IRD), Universite Cote d'Azur, Universite D'Aix-Marseille, Universite De Montpellier. The grantee listed for this patent is Centre National de la Recherche Scientifique (CNRS), Ecole Nationale Superieure de Chimie, Institut de Recherche Pour le Developpement (IRD), Universite Cote d'Azur, Universite D'Air-Marseille, Universite De Montpellier. Invention is credited to Nicolas Blondeau, Celine Crauste, Thierry Durand, Francisco Veas, Joseph Vercauteren.
United States Patent |
11,419,841 |
Crauste , et al. |
August 23, 2022 |
Uses of lipophenolic compounds
Abstract
The present invention relates to compound of formula (I):
##STR00001## wherein R is O--R.sub.3 or ##STR00002## R.sub.1 and
R.sub.2 are identical or different and are each independently H,
(C.sub.1-C.sub.6)alkyl, --CO--(C.sub.1-C.sub.21)alkyl or
--CO--(C.sub.11-C.sub.21)alkenyl group, provided that at least one
of R.sub.1 or R.sub.2 is H or (C.sub.1-C.sub.6)alkyl, R.sub.3 is a
--CO--(C.sub.11-C.sub.21)alkyl or --CO--(C.sub.11-C.sub.21)alkenyl
group, or its pharmaceutically acceptable salts, racemates,
diastereoisomers, enantiomers, or mixtures thereof, for use in
prevention and/or treatment of a disease or disorder linked to an
exacerbated vascular, lymphatic or mucosal permeability.
Inventors: |
Crauste; Celine (Montpellier,
FR), Vercauteren; Joseph (Castelnau le Lez,
FR), Veas; Francisco (Mauguio, FR), Durand;
Thierry (Montpellier, FR), Blondeau; Nicolas
(Nice, FR) |
Applicant: |
Name |
City |
State |
Country |
Type |
Universite De Montpellier
Centre National de la Recherche Scientifique (CNRS)
Ecole Nationale Superieure de Chimie
Institut de Recherche Pour le Developpement (IRD)
Universite D'Air-Marseille
Universite Cote d'Azur |
Montpellier
Paris
Montpellier
Marseilles
Marseilles
Nice |
N/A
N/A
N/A
N/A
N/A
N/A |
FR
FR
FR
FR
FR
FR |
|
|
Assignee: |
Universite De Montpellier
(N/A)
Centre National de la Recherche Scientifique (CNRS)
(N/A)
Ecole Nationale Superieure de Chimie (N/A)
Institut de Recherche Pour le Developpement (IRD) (N/A)
Universite D'Aix-Marseille (N/A)
Universite Cote d'Azur (N/A)
|
Family
ID: |
1000006515768 |
Appl.
No.: |
16/762,739 |
Filed: |
November 12, 2018 |
PCT
Filed: |
November 12, 2018 |
PCT No.: |
PCT/EP2018/080915 |
371(c)(1),(2),(4) Date: |
May 08, 2020 |
PCT
Pub. No.: |
WO2019/092239 |
PCT
Pub. Date: |
May 16, 2019 |
Prior Publication Data
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|
|
Document
Identifier |
Publication Date |
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US 20200360334 A1 |
Nov 19, 2020 |
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Foreign Application Priority Data
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Nov 10, 2017 [EP] |
|
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17306560 |
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K
31/235 (20130101) |
Current International
Class: |
A61K
31/235 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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9323075 |
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Nov 1993 |
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WO |
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2005069998 |
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Aug 2005 |
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WO |
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2006123178 |
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Nov 2006 |
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WO |
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2015162265 |
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Oct 2015 |
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WO |
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Other References
Chen et al. (Acta Tropica, 173, 2017, 76-84) (Year: 2017). cited by
examiner .
Berge et al., Pharmaceutical Salts, Journal of Pharmaceutical
Sciences, Jan. 1977, pp. 1-19, vol. 66, No. 1. cited by applicant
.
Blasi et al., Immortalization of murine microglial cells by a
v-raf/v-myc carrying retrovirus, Journal of Neuroimmunology, May
1990, pp. 229-237, vol. 27, Elsevier. cited by applicant .
Calabriso et al., Red Grape Skin Polyphenols Blunt Matrix
Metalloproteinase-2 and 9 Activity and Expression in Cell Models of
Vascular Inflammation: Protective Role in Degenerative and
Inflammatory Diseases, Molecules, Aug. 2016, 18 pages, 1147, vol.
21, XP055454556. cited by applicant .
Chang et al., Development of a Solid Dispersion System for
Improving the Oral Bioavailability of Resveratrol in Rats, Eur J
Drug Metab Pharmacokinet, published online Apr. 2016, 11 pages.
cited by applicant .
Cheng et al., Resveratrol inhibits MMP-9 expression by
up-regulating PPAR a expression in an oxygen glucose
deprivation-exposed neuron model, Neuroscience Letters, Feb. 2009,
pp. 105-108, vol. 451, No. 2. cited by applicant .
Crauste et al., Synthesis and Evaluation of Polyunsaturated Fatty
Acid-Phenol Conjugates as Anti-Carbonyl-Stress Lipophenols,
European Journal of Organic Chemistry, Jul. 2014, pp. 4548-4561 (15
pages). cited by applicant .
Delaunay et al., Preparative isolation of polyphenolic compounds
from Vitis vinifera by centrifugal partition chromatography,
Journal of Chromatography A, Jul. 2002, pp. 123-128, vol. 964,
Elsevier. cited by applicant .
Fulgenzi et al., In Vivo Inhibition of TNFa-lnduced Vascular
Permeability by Resveratrol, Transplantation Proceedings, May 2001,
pp. 2341-2343, vol. 33, XP055454461. cited by applicant .
Gao et al., Resveratrol protects primary cortical neuron cultures
from transient oxygen-glucose deprivation by inhibiting MMP-9,
Molecular Medicine Reports, published online Mar. 2014, pp.
2197-2204, vol. 9, No. 6. cited by applicant .
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dated Jan. 28, 2019, pp. 1-4. cited by applicant .
Lee et al., Resveratrol inhibits TNF-a-induced proliferation and
matrix metalloproteinase expression in human vascular smooth muscle
cells, The Journal of Nutrition, Dec. 2005, pp. 2767-2773, vol.
135, No. 12, American Society for Nutrition, XP009503755. cited by
applicant .
Luplerdlop et al., Dengue-virus-infected dendritic cells trigger
vascular leakage through metalloproteinase overproduction, EMBO
Reports, Nov. 2006, pp. 1176-1181, vol. 7, No. 11. cited by
applicant .
Marsac et al., Infection of human monocyte-derived dendritic cells
by ANDES Hantavirus enhances proinflammatory state, the secretion
of active MMP-9 and indirectly enhances endothelial permeability,
Virology Journal, May 2011, pp. 1-9, vol. 8, No. 223. cited by
applicant .
Misse et al., HIV-1 glycoprotein 120 induces the MMP-9
cytopathogenic factor production that is abolished by inhibition of
the p38 mitogen-activated protein kinase signaling pathway, Blood,
Aug. 2001, pp. 541-547, vol. 98, No. 3. cited by applicant .
Mosmann, Rapid Colorimetric Assay for Cellular Growth and Survival:
Application to Proliferation and Cytotoxicity Assays, Journal of
Immunological Methods, Dec. 1983, pp. 55-63, vol. 65, Elsevier.
cited by applicant .
Pany et al., PKC Activation by Resveratrol Derivatives with
Unsaturated Aliphatic Chain, PLoS ONE, Dec. 2012, pp. 1-11, vol. 7,
No. 12, e52888, XP055454605. cited by applicant .
Shamseddin et al., Resveratrol-Linoleate protects from exacerbated
endothelial permeability via a drastic inhibition of the MMP-9
activity, Bioscience Reports, Jul. 2018, pp. 1-13, vol. 38. No. 4,
BSR20171712, XP055540761. cited by applicant .
Walle, Bioavailability of resveratrol, Annals of the New York
Academy of Sciences, Jan. 2011, pp. 9-15, vol. 1215. cited by
applicant.
|
Primary Examiner: Ramachandran; Umamaheswari
Attorney, Agent or Firm: Lerner, David, Littenberg, Krumholz
& Mentlik, LLP
Claims
The invention claimed is:
1. A method for the treatment of encephalitis comprising
administering, to a person in need thereof, a compound of formula
(IIb): ##STR00064## with R.sub.3 selected from the group consisting
of: ##STR00065## or its pharmaceutically acceptable salts,
racemates, diastereoisomers, enantiomers, or mixtures thereof.
2. The method of claim 1, wherein the compound is administered for
1 to 4 days, and just before and/or during the acute phase response
associated with factors inducing the increased vascular, lymphatic
or mucosal permeability.
3. The method of claim 1, wherein the compound of formula (IIb) is
administered in association with at least one additional active
compound selected from the group consisting of antibiotic,
antiviral, antifungal, antiparasitic, anti-inflammatory active
compound and mixtures thereof.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
The present application is a national phase entry under 35 U.S.C.
.sctn. 371 of International Application No. PCT/EP2018/080915 filed
Nov. 12, 2018, which claims priority from EP Application No.
17306560.8 filed Nov. 10, 2017, all of which are incorporated
herein by reference.
TECHNICAL DOMAIN
The present invention concerns new uses of lipophenolic compounds,
in particular for the prevention and/or treatment of diseases or
disorders linked to an exacerbated vascular, lymphatic or mucosal
permeability, in particular a chronic or acute disease or disorder,
preferably an acute inflammatory disorder.
BACKGROUND OF THE INVENTION
During evolution, the defense mechanisms of organisms (innate
immunity) have been strongly selected, therefore highly conserved.
In this context, inflammation is a process by which organisms
respond to attacks from the `non-self` (including pathogens,
environment, UV radiation, toxic or food, immunity) to maintain the
integrity in homeostatic conditions. Inflammation can be due to a
variety of reasons or a combination of them including cancer,
trauma, infection (HIV, HTLV-1), auto-immunity having serious
consequences such as epilepsy and diabetes, viral hemorrhagic
fevers (Dengue, Ebola, Hantavirus, Marbug, Lassa), etc.
One of the major consequences of the inflammatory processes is the
pathological expression of an increased vascular permeability
(IVP), also named exacerbated vascular permeability or enhanced
vascular permeability. The permeability according to the invention
also encompasses lymphatic or mucosal permeability.
It is known that the activation of target cells such as monocytes
(dendritic cells, macrophages) by viruses, including human
immunodeficiency virus (HIV), Dengue hemorrhagic virus (DENV) and
Hantavirus cardiopulmonary hemorrhage (ANDES-HV) lead to expression
of mediators involved in inflammatory process, resulting in the
production of proteases, in particular matrix metalloproteases,
involved in increased vascular permeability (IVP) (Misse D. et al.,
2001; Marsac D. et al., 2011 and Luplertlop N. el al., 2006).
Both MMP-2 and MMP-9, also known as gelatinases, play an important
role in several pathological disorders including pulmonary
infections, cancer, bronchial asthma, chronic wounds and
inflammation, malaria, multiple sclerosis, rheumatoid arthritis,
cardiovascular and central nervous system disorders. In particular,
MMP-9 is known to destroy the basal lamina, disrupt blood brain
barrier (BBB) endothelium and cause an extravasation of phagocytes
and parasite-infected erythrocytes, suggesting a main role in
Cerebral Malaria CM. In addition, several clinical studies have
identified relationships between elevated levels of proinflammatory
cytokines (11-b, IL-6, TNF-a), and stroke-induced brain injury.
Upon reaching critical levels, these proinflammatory factors
contribute to the evolution of tissue injury by two main pathways.
They may exert direct cytotoxic effects, like TNF-.alpha. (Tumor
Necrosis Factor-alpha), threatening neuronal viability in the
penumbra or indirect effects promoting leukocyte transmigration
across the blood-brain barrier (BBB) that, in consequence, feeds
inflammatory cascades, release of oxygen-free radicals and
proteolytic enzymes like matrix metalloproteinase-9 (MMP-9)
mediating BBB breakdown.
MMP-9 is also over produced in immature dendritic cells infected
with dengue virus, and this over activity is associated with
elevated level of endothelial permeability (Luplertlop et al.
2006).
So, there is still a need to develop new selective MMP inhibitors
for therapeutic applications, in particular MMP-9 inhibitors for
preventing and/or treating diseases or disorders linked to an
exacerbated (increased) vascular, lymphatic or mucosal
permeability.
By `MMP-9 inhibitors` according to the invention, it means
inhibitors of the activity of MMP-9; it can be compounds that exert
their effect on the MMP9 activity via the enzymatic activity,
expression, post-translational modifications or by other means.
In particular, the Applicant has developed new inhibitors of the
activity of MMP-9 for preventing and/or treating diseases or
disorders linked to an exacerbated vascular, lymphatic or mucosal
permeability, in particular induced and/or aggravated by infectious
agents including virus, such as viral hemorrhagic fevers, in
particular the ones caused by Dengue or Ebola virus.
Dengue virus (DENV) is classified by the World Health Organization
into three categories; dengue with warning signs, dengue without
warning signs, and severe dengue. The severe symptoms appearing
with an incubation period between 3-8 days, start by sudden fever
that lasts between a few days to one week, and the fever is usually
associated with headache, myalgia, arthralgia, skin rash, retro
ocular pain, nausea and vomiting. The critical phase usually starts
after 48 hours when the fever goes down, vascular permeability
increases with pleural leakage, ascites, mucosal bleeding, elevated
level of hematocrit and shock. In the recovery period, the
extravascular fluid is gradually reabsorbed during the treatment
followed by secondary skin rash and fatigue that may last for
several weeks.
The mechanism of pathogenesis starts when the virus is injected in
host after a mosquito bite, the introduced viruses target immune
cells monocytes, macrophages and dendritic cells through mannose
receptors and dendritic cells-specific ICAM-3-grabbing non-integrin
1 (DC-SIGN), respectively. The infected dendritic cell migrates to
the lymph nodes so that it can present the dengue antigen to the T
lymphocytes. Once the virions are released, they can infect other
immune cells such as monocytes, macrophages and lymphocytes. The
virus then can move from lymph nodes to blood through the infected
lymphocytes causing further infection and replication that leads to
viremia. Antigens presented on major histocompatibility complex
(MHC) molecules on the surface of the infected cells activate
several types of immune cells, for instance they stimulate the
production of IL-2, IL-4, IL-5 and IL-6 by CD4+ and CD8+ cells,
IL-1, IL-6 and TNF-.alpha. by macrophages and TNF-.alpha. and
IL-1.beta. by monocytes. All the above-mentioned cytokines and
immune mediators serve to increase the vascular permeability,
hemorrhage and worsen the prognosis of the disease.
The present invention aims to provide lipophenolic compounds, in
particular lipophenolic derivatives of resveratrol, for which the
inventors demonstrated an improved inhibition activity of MMP-9 in
comparison to resveratrol as such.
Resveratrol, a natural polyphenolic phytoalexin compound that
exists in grape stalks, nuts and other plants, has been reported to
inhibit the activity of MMP-9 (Cheng et al. 2009, Gao et al. 2014).
It also has several biological activities including anti-oxidant,
anti-inflammatory and anti-viral activities. Regardless of the
above mentioned in vitro biological activities, its use in in vivo
treatment is still not considered due to its poor bioavailability
(Walle 2011, Chang et al. 2016).
The inventors have now developed lipophenolic derivatives with
fatty acid linked to the resorcinol cycle in position 3 (cycle A),
or linked to the cycle B in position 4', and tested their ability
to inhibit activity of MMP-9 in activated THP-1 cell line. They
found that the position of lipids on the resveratrol scaffold
structure has an impact on the inhibition activity of MMP9. In
particular, 3-Resv-DHA with fatty acid linked to the resorcinol
cycle in position 3 did not show any inhibitory activity compare to
its region-isomer, 4'-Resv-DHA. All together, these findings show
that the resorcinol moiety seems to confer a "structure-activity"
relation of the compound. Moreover, saturated fatty-acid are much
less flexible than polyunsaturated fatty acids, and the difference
in the activity between saturated, DHA (6 double bonds) and LA (2
double bonds) conjugates could have an impact on the affinity of
the active site targeted and involved in the inhibition activity of
MMP9, the omega-6 linoleic acid incorporated at the 4' position
(Resv-LA), giving in the present invention the best activity
compared to Resv-DHA and Resv-C22 (Resv-BE).
SUMMARY OF THE INVENTION
A first object of the invention is a compound of formula (I):
##STR00003## wherein R is O--R.sub.3 or
##STR00004## R.sub.1 and R.sub.2 are identical or different and are
each independently H, (C.sub.1-C.sub.6)alkyl,
--CO--(C.sub.1-C.sub.21)alkyl or --CO--(C.sub.11-C.sub.21)alkenyl
group, provided that at least one of R.sub.1 or R.sub.2 is H or
(C.sub.1-C.sub.6)alkyl, R.sub.3 is a --CO--(C.sub.11-C.sub.21)alkyl
or --CO--(C.sub.11-C.sub.21)alkenyl group, or its pharmaceutically
acceptable salts, racemates, diastereoisomers, enantiomers, or
mixtures thereof, for use in prevention and/or treatment of a
disease or disorder linked to an exacerbated (increased) vascular,
lymphatic or mucosal permeability, in particular a chronic or acute
disease or disorder, preferably an acute inflammatory disorder.
The term `diseases` and `disorders` are used interchangeably in the
present disclosure.
The term `treatment` or `treating` according to the invention means
reversing, alleviating, inhibiting the progress of, or preventing
the disorder or condition to which such term applies, or one or
more symptoms of such disorder or condition.
The inventors demonstrated that the lipophenolic compounds
according to the invention are able to protect the endothelial
barrier integrity and decrease the exacerbated (increased)
vascular, lymphatic or mucosal permeability in infections and other
diseases.
The expression `vascular permeability` according to the invention
encompasses endothelial and/or epithelial permeability of the
vascular system (i.e cardiovascular system). This expression is
used interchangeably with `vascular leakage` or `endothelial and/or
epithelial exacerbated permeability`.
The present invention also encompasses `lymphatic permeability`
which comprises endothelial and/or epithelial permeability of the
vessels of the lymphatic system.
The present invention also encompasses `mucosal permeability` which
comprises endothelial and/or epithelial permeability of the mucous
(ex: genital, pulmonar, or digestive system).
So, the generic expression `permeability` according to the
invention encompasses vascular, lymphatic or mucosal
permeability.
The expression `exacerbated or increased vascular permeability` in
the present invention means in particular any abnormal (i.e:
pathological) increase (or enhancement) of vascular, lymphatic or
mucosal permeability associated in particular with inflammation
process.
The exacerbated (increased) vascular or lymphatic permeability
according to the invention generally results from local (i.e.,
organs, tissues) or general homeostasis disruption.
The homeostasis is commonly known and may be defined as a tendency
of an organism, a cell, an organ or a tissue, to regulate its
internal conditions, usually by a system of feedback controls, so
as to stabilize health and functioning, regardless of the outside
changing conditions.
Such homeostasis disruption can be observed during an exacerbated
acute phase immune response (inflammation resulting from a major
aggressive cause) or during a chronic inflammatory process. The
main actors of such deleterious effects on vascular, lymphatic or
mucosal dysfunctions include infectious agents, trauma, allergy,
cardiovascular disorders, central nervous system disorders,
autoimmune diseases, metabolic diseases and mixtures thereof.
By `disease or disorder linked to an increased or exacerbated
permeability`, it means a disease or disorder induced and/or
amplified (aggravated) by an increased vascular, lymphatic or
mucosal permeability.
In particular, it results from the expression of chronic
inflammation or of intense acute phase inflammation observed in
particular during chronic or acute conditions, in particular during
infectious processes caused by pathogens including viruses.
In a particular embodiment, the disease or disorder is a chronic
disease or disorder.
`Chronic` conditions or disorders develop slowly and may worsen
over an extended period of time--months to years. They are often
caused by unhealthy behaviors that increase the risk of
disease--poor nutrition, inadequate physical activity, overuse of
alcohol, or smoking. Social, emotional, environmental, and genetic
factors also play a role. As people age, they are more likely to
develop one or more chronic conditions. The chronic conditions are
slower to develop, may progress over time, and may have any number
of warning signs or no signs at all. Unlike acute conditions, some
chronic health conditions cannot be cured--only controlled.
In another particular and preferred embodiment, the disease or
disorder is an acute disease or disorder.
`Acute` conditions or disorders generally develop suddenly and last
a short time, often only a few days or weeks. They are often caused
by a virus or an infection, but can also be caused by an injury
resulting from a trauma as example. They come on rapidly, and are
accompanied by distinct symptoms that require urgent or short-term
care, and get better once they are treated.
In a particular embodiment, the lipophenolic compounds are for use
in prevention and/or treatment of an acute condition, in particular
an acute inflammation disorder (acute inflammatory disorder).
An inflammation (inflammation disorder) is an immune reaction that
results in localized redness, warmth and swelling. It generally
occurs in response to an infection, irritation or injury.
In particular, the disease or disorder linked to an increased or
exacerbated permeability is selected from chronic and acute
condition, preferably acute condition and in particular infectious
diseases, allergies, trauma, cardiovascular disorders, central
nervous system disorders, auto-immune diseases, metabolism
diseases, or mixtures thereof, which may be induced and/or
aggravated by infectious agents.
In a particular embodiment, the lipophenolic compound used
according to the invention is administered for a short-term
treatment, in particular from 1 to 4 days, and preferably just
before and/or during the acute phase response associated with
factors inducing the increased vascular, lymphatic or mucosal
permeability, including MMP-9. In a particular embodiment, the
lipophenolic compound used according to the invention is used in
association with at least one additional active compound selected
from the group consisting of antibiotic, antiviral, antifungal,
antiparasitic, anti-inflammatory active compound and mixtures
thereof.
The invention also concerns specific compounds of formula IIb
(resveratrol derivatives), pharmaceutical compositions and
pharmaceutical kits comprising them.
DETAILED DESCRIPTION OF THE INVENTION
The present invention concerns a compound of formula (I):
##STR00005## wherein R is O--R.sub.3 or
##STR00006## R.sub.1 and R.sub.2 are identical or different and are
each independently H, (C.sub.1-C.sub.6)alkyl,
--CO--(C.sub.1-C.sub.21)alkyl or --CO--(C.sub.11-C.sub.21)alkenyl
group, provided that at least one of R.sub.1 or R.sub.2 is H or
(C.sub.1-C.sub.6)alkyl, R.sub.3 is a --CO--(C.sub.11-C.sub.21)alkyl
or --CO--(C.sub.11-C.sub.21)alkenyl group, or its pharmaceutically
acceptable salts, racemates, diastereoisomers, enantiomers, or
mixtures thereof, for use in prevention and/or treatment of a
disease or disorder linked to an exacerbated (increased) vascular,
lymphatic or mucosal permeability.
The compounds used in the invention are lipophenolic compounds and
thus phenolic derivatives. They comprise a phenyl group which may
carry one or several phenol functions, preferably two (resveratrol
derivatives), each being possibly alkylated or acylated. Each
compound according to the invention comprises at least one lipidic
chain (also named fatty acid chain) which corresponds to the
R.sub.3 radical. The compounds used in the invention may also be
called fatty acid-phenolic conjugates or derivatives as they
comprise a phenolic core on which is linked at least one fatty acid
chain. In a preferred embodiment, for the resveratrol derivatives
(formula IIb disclosed hereunder), the fatty acid chain is on
position 4'. The inventors demonstrated that such resveratrol
compounds with fatty acid chain on position 4' have a MMP-9
inhibitory activity, whereas resveratrol derivatives with fatty
acid chain in position 3 do not have any detectable MMP-9
inhibitory activity.
The hydrophilic/lipophilic balance is adjusted by a covalent
grafting of a lipid molecule (a fatty acid) on the phenol
structure, preferably the resveratrol, and designs new lipophenolic
derivatives. These resveratrol-linked fatty acids or fatty-acid
resveratrol derivatives include in particular saturated fatty acid
(docosanoic acid or behenic acid, C22:0, Resv-C22 also named
Resv-BE), omega-6 polyunsaturated fatty acid using linoleic acid
(C18:2 n-6, Resv-LA), and omega-3 polyunsaturated fatty acid such
as docosahexaenoic acid (C22:6 n-3, Resv-DHA) and linolenic acid
(C18:3 n-3, Resv-ALA).
The term "an alkyl group" according to the invention means a linear
or branched, saturated, hydrocarbon-based aliphatic group
comprising, unless otherwise mentioned, from 1 to 12 carbon atoms.
By way of examples, mention may be made of methyl, ethyl, n-propyl,
isopropyl, butyl, isobutyl, tert-butyl or pentyl groups.
The term "alkenyl" according to the invention includes partially
unsaturated, nonaromatic, hydrocarbon groups.
The term "pharmaceutically acceptable salts" refers to salts which
retain the biological effectiveness and properties of the compounds
of the invention and which are not biologically or otherwise
undesirable. Pharmaceutically acceptable acid addition salts may be
prepared from inorganic and organic acids, while pharmaceutically
acceptable base addition salts can be prepared from inorganic and
organic bases. For a review of pharmaceutically acceptable salts
see Berge, et al. ((1977) "Pharmacologically acceptable salts" J.
Pharm. Sd, vol. 66, 1). For example, the salts include those
derived from inorganic acids such as hydrochloric, hydrobromic,
sulfuric, sulfamic, phosphoric, nitric, and the like, as well as
salts prepared from organic acids such as acetic, propionic,
succinic, glycolic, stearic, lactic, malic, tartaric, citric,
ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic,
benzoic, salicyclic, sulfanilic, fumaric, methanesulfonic, and
toluenesulfonic acid and the like.
In a particular embodiment, the radical R in the compound of
formula (I)
##STR00007## is O--R.sub.3.
Such lipophenolic compounds, also named `fatty-acid phloroglucinol
derivatives` have formula (Ia), and preferably formula (Ib) as
disclosed hereunder:
##STR00008##
The radicals R.sub.1, R.sub.2 and R.sub.3 are defined above and in
the following description.
In a particular and preferred embodiment, the radical R in the
compound of formula (I)
##STR00009## is
##STR00010##
Such lipophenolic compounds, also named `fatty-acid resveratrol
derivatives` have formula (IIa), and preferably formula (IIb) as
disclosed hereunder:
##STR00011##
The radicals R.sub.1, R.sub.2 and R.sub.3 are defined above and in
the following description.
In a particular embodiment, the radicals R.sub.1, R.sub.2 and
R.sub.3 in the compound of formula (I)
##STR00012## wherein R is O--R.sub.3 or
##STR00013## are respectively: R.sub.1 is R.sub.3 and R.sub.2 is H
or (C.sub.1-C.sub.6)alkyl, or R.sub.2 is R.sub.3 and R.sub.1 is H
or (C.sub.1-C.sub.6), alkyl.
In a particular embodiment, R.sub.1 and R.sub.2 are both H.
In a particular and preferred embodiment, the radical R in the
compound of formula (I)
##STR00014## is
##STR00015## and R.sub.1 and R.sub.2 are both H, corresponding to
the compound of formula (IIb)
##STR00016##
In a particular embodiment, R.sub.3 is a linear
--CO--(C.sub.11-C.sub.21) alkyl or --CO--(C.sub.11-C.sub.21)
alkenyl group.
In particular, R.sub.3 is a linear --CO--(C.sub.15-C.sub.21) alkyl
chain, with said alkyl chain preferably containing an uneven number
of carbon atoms, or a linear --CO--(C.sub.15-C.sub.21)alkenyl
chain, with said alkenyl chain preferably containing an uneven
number of carbon atoms and advantageously cis double bond(s).
In a preferred embodiment, R.sub.3 is selected from the group
consisting of:
##STR00017## also named docosanoic (C.sub.22) or behenic acid (BE)
derivative,
##STR00018## also named linolenic acid (ALA) derivative
##STR00019## also named linoleic acid (LA) derivative or
##STR00020## also named docosahexanoic acid (DHA) derivative
preferably
##STR00021## and more preferably
##STR00022##
In a particular and preferred embodiment, lipophenolic compound
used in the invention is a compound of formula (IIb), also named
fatty-acid resveratrol derivative in position 4'
##STR00023## with R.sub.3 selected from the group consisting
of:
##STR00024## also named Resv-C.sub.22 or Resv-BE
##STR00025## also named Resv-ALA
##STR00026## also named Resv-LA or
##STR00027## also named Resv-DHA preferably
##STR00028## and more preferably
##STR00029##
In a more preferred embodiment, the lipophenolic compound used in
the invention is a compound of formula (IIb),
##STR00030## with R.sub.3 being
##STR00031## also named Resv-LA.
The lipophenolic compounds as disclosed above are used according to
the invention to protect the endothelial barrier integrity and
decrease the exacerbated (increased) vascular, lymphatic or mucosal
permeability, which generally results from local (i.e., organs,
tissues) or general homeostasis disruption.
As disclosed above, the main actors of such deleterious effects on
vascular, lymphatic or mucosal dysfunctions include infectious
agents, trauma, allergy, cardiovascular disorders, central nervous
system disorders, autoimmune diseases, metabolic diseases and
mixtures thereof.
So, the disease or disorder linked to an exacerbated vascular,
lymphatic or mucosal permeability according to the invention is in
particular selected from chronic and acute condition, preferably
acute condition, and in particular infectious diseases, trauma,
allergy, cardiovascular disorders, central nervous system
disorders, autoimmune diseases, metabolic diseases and mixtures
thereof, which may be induced and/or aggravated by infectious
agents.
In particular, the disease or disorder linked to exacerbated
vascular, lymphatic or mucosal permeability is induced and/or
aggravated by infectious agents, in particular virus, and is
selected preferably from central nervous system disorders,
auto-immune diseases and viral infections, more preferably from
viral hemorrhagic fevers and in particular the ones caused by
Dengue or Ebola virus.
In a particular embodiment, the lipophenolic compounds as disclosed
above are used according to the invention to decrease the
exacerbated (increased) vascular, lymphatic or mucosal permeability
induced and/or aggravated by infectious agents causing infectious
diseases.
In another embodiment, the lipophenolic compounds as disclosed
above are used according to the invention to decrease the
exacerbated (increased) vascular, lymphatic or mucosal permeability
induced and/or aggravated by a trauma.
The term `trauma` according to the invention refers to injury or
damage to a biological organism caused by physical harm from an
external source. Mentioned may be made in particular of
cardiovascular event or `stroke` or injuries caused during combat
sports (e.g boxing) or contact sports (e.g rugby).
In another embodiment, the lipophenolic compounds as disclosed
above are used according to the invention to decrease the
exacerbated (increased) vascular, lymphatic or mucosal permeability
induced and/or aggravated by an allergy.
In another embodiment, the lipophenolic compounds as disclosed
above are used according to the invention to decrease the
exacerbated (increased) vascular, lymphatic or mucosal permeability
induced and/or aggravated by a cardiovascular disorder. Mention may
be made in particular of heart failure, cardiovascular event or
stroke.
In another embodiment, the lipophenolic compounds as disclosed
above are used according to the invention to decrease the
exacerbated (increased) vascular, lymphatic or mucosal permeability
induced and/or aggravated by central nervous system disorder.
Mention may be made in particular of encephalitis, epilepsy.
In another embodiment, the lipophenolic compounds as disclosed
above are used according to the invention to decrease the
exacerbated (increased) vascular, lymphatic or mucosal permeability
induced and/or aggravated by auto-immune diseases. Mention may be
made in particular of multiple sclerosis.
In another embodiment, the lipophenolic compounds as disclosed
above are used according to the invention to decrease the
exacerbated (increased) vascular, lymphatic or mucosal permeability
induced and/or aggravated by metabolic diseases. Mention may be
made in particular of diabetes.
In a particular preferred embodiment, the lipophenolic compounds
are used to decrease the exacerbated (increased) vascular,
lymphatic or mucosal permeability induced and/or aggravated by
infectious agents (e.g virus, bacteria, parasite or fungus).
In a first preferred embodiment, the lipophenolic compounds are
used to decrease the exacerbated (increased) vascular, lymphatic or
mucosal permeability induced and/or aggravated by an infectious
agent causing an inflammation of the central nervous system also
named `central nervous system disorder`.
The term `inflammation of the central nervous system` or `central
nervous system disorder` according to the invention, refers the
inflammation of the brain (encephalitis) and the spinal cord as
well as the tissues that protect the brain called meninges.
Encephalitis with meningitis is known as meningoencephalitis.
General symptoms include headache, fever, confusion, dizziness
drowsiness, fatigue, reaching seizures or convulsions, tremors,
hallucinations, stroke, and/or memory dysfunctions.
Inflammation of the nervous system, and in particular encephalitis
can be caused by viral infection (e.g.: rabies virus, HSV
infection, poliovirus, and measles virus may cause acute viral
encephalitis such as Japanese encephalitis virus, Nipah virus, Zika
virus, Hendra virus, West Nile virus, Herpes simplex virus etc) or
bacterial infection (e.g.: bacterial meningitis); parasitic
infection (e.g.: toxoplasma, malaria, etc) as well as fungi (e.g.:
cryptococcidiosis, candidiasis, aspergillosis). In another hand, as
disclosed above, encephalitis may be caused by inflammatory
cascades: proinflammatory factors (cytokines), upon reaching
critical levels, contribute to the evolution of tissue injury
either by exerting direct cytotoxic effects, like TNF-.alpha.
threatening neuronal viability in the penumbra or indirect effects
promoting leukocyte transmigration across the blood-brain barrier
(BBB) that, in consequence, feeds inflammatory cascades, release of
oxygen-free radicals and proteolytic enzymes like matrix
metalloproteinase-9 (MMP-9) mediating BBB breakdown and nervous
system disorders, in particular encephalitis.
In a second preferred embodiment, the lipophenolic compounds are
used to decrease the exacerbated (increased) vascular, lymphatic or
mucosal permeability induced and/or aggravated by a virus causing a
viral hemorrhagic fever.
The most evident examples of this exacerbated vascular leakage are
induced by viruses causing hemorrhagic fevers (VHFs), which is a
diverse group of animal and human illnesses (e.g: RNA viruses: the
families Arenaviridae, Filoviridae, Bunyaviridae, Flaviviridae, and
Rhabdoviridae). All types of VHF are characterized by fever and
bleeding disorders and all can progress to high fever, shock and
death in most of the cases. Some of the VHF agents cause relatively
mild illnesses, such as the Scandinavian nephropathia epidemica (a
Hantavirus), while others, such as Ebola, Lassa, Crimean-Congo
hemorrhagic fever, CCHF, Marburg viruses, and the South American
hemorrhagic fevers caused by arenaviruses most of the times cause
severe, life-threatening disease, only observed in a small
percentage of patients with Dengue, Rift Valley or Lassa virus
infection.
Five families of RNA viruses have been recognized as being able to
cause hemorrhagic fevers: Arenaviridae: include the viruses
responsible for Lassa fever (Lassa virus), Lujo virus, Argentine
(Junin virus), Bolivian (Machupo virus), Brazilian (Sabia virus),
Chapare hemorrhagic fever (Chapare virus) and Venezuelan (Guanarito
virus) hemorrhagic fevers; Bunyaviridae: include the members of the
Hantavirus genus that cause hemorrhagic fever with renal syndrome
(HFRS), the Crimean-Congo hemorrhagic fever (CCHF) virus from the
Nairovirus genus, Garissa virus and Ilesha virus from the
Orthobunyavirus and the Rift Valley fever (RVF) virus from the
Phlebovirus genus; Filoviridae: include Ebola virus and Marburg
virus; Flaviviridae: include dengue, yellow fever, and two viruses
in the tick-borne encephalitis group that cause VHF: Omsk
hemorrhagic fever virus and Kyasanur Forest disease virus; and
Rhabdoviridae.
In particular, the prevention and/or treatment of the disease or
disorder according to the invention resulted from the reduction, by
using lipophenolic compounds of formula (I) according to the
invention, of the active MMP9-induced endothelial or epithelial
hyper-permeability.
In particular embodiments, the lipophenolic compounds of formula
(I) according to the inventions, ie fatty-acid phloroglucinol
derivatives, are used for prevention and/or treatment of diseases
as defined above and listed in the following Table 1:
TABLE-US-00001 Central Infectious Auto- nervous diseases, Viral
Trauma immune system in hemorrhagic (ex: diseases Cardiovascular
Metabolic Fatty-acid phloroglucinol disorders particular fevers
(ex: stroke, (ex: diseases (ex: diseases derivatives according to
(ex: caused by Dengue, combat multiple heart failure, (ex: formula
(I) encephalitis) a virus Ebola) Allergies sports) sclerosis)
stroke) diabete) ##STR00032## x xx xxx xx xx x x x R is O--R.sub.3
R.sub.1 and R.sub.2 are identical or different and are each
independently H, (C.sub.1- C.sub.6)alkyl, --CO--(C.sub.1-
C.sub.21)alkyl or --CO--(C.sub.11-C.sub.21)alkenyl group, provided
that at least one of R.sub.1 or R.sub.2 is H or
(C.sub.1-C.sub.6)alkyl, R.sub.3 is a --CO--(C.sub.11-C.sub.21)alkyl
or --CO--(C.sub.11-C.sub.21)alkenyl group Wherein R1 and R2 = H xxx
xx xxx xx xxx xxx xx xx Wherein R1 and R2 = H xxx xx xxx xx xxx xxx
xx xx and R3 is a linear --CO--(C.sub.11-C.sub.21)alkyl or
--CO--(C.sub.11-C.sub.21)alkenyl group, preferably
--CO--(C.sub.15-C.sub.21)alkyl or --CO--(C.sub.15-C.sub.21)alkenyl
group. Wherein R1 and R2 = H xxx xx xxx xx xxx xxx xx xx and R3 is
--CO--(C.sub.22)alkyl group Wherein R1 and R2 = H xxx xx xxx xx xxx
xxx xx xx and R3 is --CO--(C.sub.18)alkenyl group with 2 double
bonds Wherein R1 and R2 = H xxx xx xxx xx xxx xxx xx xx and R3 is
--CO--(C.sub.22)alkenyl group with 6 double bonds
All the combinations of fatty-acid phloroglucinol derivatives with
diseases disclosed above in the table 1 are encompassed by the
present invention. The combinations `xx` are preferred and the
combinations `xxx` are more preferred.
In particular and preferred embodiments, the lipophenolic compounds
of formula (IIa) and (IIb) according to the inventions, ie
fatty-acid resveratrol derivatives, are used for the prevention
and/or treatment of diseases as defined above and listed in the
following Table 2:
TABLE-US-00002 Central Infectious Auto- nervous diseases, Viral
Trauma immune system in hemorrhagic (ex: diseases Cardiovascular
Metabolic disorders particular fevers (ex: stroke, (ex: diseases
(ex: diseases Fatty-acid resveratrol derivatives (ex: caused by
Dengue, combat multiple heart failure, (ex: according to formula
(II) encephalitis) a virus Ebola) Allergies sports) sclerosis)
stroke) diabete) ##STR00033## xxx xx xxx xx xxx xxx xx xx
##STR00034## R.sub.1 and R.sub.2 are identical or different and are
each independently H, (C.sub.1-C.sub.6)alkyl,
--CO--(C.sub.1-C.sub.21)alkyl or --CO--(C.sub.11-C.sub.21)alkenyl
group, provided that at least one of R.sub.1 or R.sub.2 is H or
(C.sub.1-C.sub.6)alkyl, R.sub.3 is a --CO--(C.sub.11-C.sub.21)alkyl
or --CO--(C.sub.11-C.sub.21)alkenyl group Wherein R1 and R2 = H xxx
xx xxx xx xxx xxx xx xx Wherein R1 and R2 = H and R3 xxx xx xxx xx
xxx xxx xx xx is a linear --CO--(C.sub.11-C.sub.21)alkyl or
--CO--(C.sub.11-C.sub.21)alkenyl group, preferably --CO--(C.sub.15-
C.sub.21)alkyl or --CO--(C.sub.15-C.sub.21)alkenyl group. Wherein
R1 and R2 = H and R3 xxx xx xxx xx xxx xxx xx xx is
--CO--(C.sub.22)alkyl group (Resv-C22 or Resv-BE) Wherein R1 and R2
= H and R3 xxx xx xxx xx xxx xxx xx xx is --CO--(C.sub.18)alkenyl
group with 2 double bonds (Resv-LA) Wherein R1 and R2 = H and R3
xxx xx xxx xx xxx xxx xx xx is --CO--(C.sub.22)alkenyl group with 6
double bonds (Resv- DHA)
All the combinations of fatty-acid resveratrol derivatives with
diseases disclosed in the table 1 are encompassed by the present
invention. The combinations `xx` are preferred and the combinations
`xxx` are more preferred.
In a particular and preferred embodiment, the lipophenolic compound
is of formula (IIb),
##STR00035## with R.sub.3 selected from the group consisting
preferably of:
##STR00036## and more preferably
##STR00037## and the disease is selected from viral infections, and
preferably viral hemorrhagic fevers, in particular the ones caused
by Dengue or Ebola virus.
In a particular and preferred embodiment, the lipophenolic compound
is of formula (IIb),
##STR00038## with R.sub.3 selected from the group consisting
preferably of:
##STR00039## and more preferably
##STR00040## and the disease is selected from auto-immune diseases,
and preferably multiple sclerosis.
In a particular and preferred embodiment, the lipophenolic compound
is of formula (IIb),
##STR00041## with R.sub.3 selected from the group consisting
preferably of:
##STR00042## and more preferably
##STR00043## and the disorder is selected from trauma, in
particular cardiovascular event or stroke or injury during combat
sports or contact sports.
In a particular and preferred embodiment, the lipophenolic compound
is of formula (IIb),
##STR00044## with R.sub.3 selected from the group consisting
preferably of:
##STR00045## and more preferably
##STR00046## and the disease is selected from central nervous
system disorder, such as encephalitis.
In a particular and preferred embodiment, the lipophenolic compound
is of formula (IIb),
##STR00047## with R.sub.3 selected from the group consisting
preferably of:
##STR00048## and more preferably
##STR00049## and the disease is selected from cardiovascular
diseases, in particular heart failure, cardiovascular event or
stroke.
Another object of the invention is a compound of formula (IIb)
##STR00050## with R.sub.3 selected from the group consisting
of:
##STR00051## preferably
##STR00052## and more preferably
##STR00053## or its pharmaceutically acceptable salts, racemates,
diastereoisomers, enantiomers, or mixtures thereof.
Another object of the invention is a pharmaceutical composition
comprising, in a pharmaceutically acceptable vehicle, at least one
compound of formula (IIb)
##STR00054## with R.sub.3 selected from the group consisting
of:
##STR00055## preferably
##STR00056## and more preferably
##STR00057## or its pharmaceutically acceptable salts, racemates,
diastereoisomers, enantiomers, or mixtures thereof.
Another object of the invention is a pharmaceutical composition or
pharmaceutical kit comprising, at least one compound of formula
(IIb) defined above in association with at least one additional
active compound selected from the group consisting of antibiotic,
antiviral, antifungal, antiparasitic, anti-inflammatory active
compound and mixtures thereof.
Galenic
The lipophenolic compound of the invention may be used as such but
is preferably formulated in a pharmaceutical composition comprising
a pharmaceutically acceptable vehicle.
In particular, the lipophenolic compound of the invention is
administered orally, intrathecally, enterally, parenterally,
nutritionally, and/or intraperitoneally.
The term "pharmaceutically acceptable" according to the invention
refers to compositions, carriers, diluents and reagents, capable of
administration to or upon a mammal, in particular a human, without
the production of undesirable physiological effects such as nausea,
gastric upset and the like.
The preparation of a pharmacological composition that contains
active ingredients dissolved or dispersed therein is well
understood in the art and need not to be limited based on
formulation. Typically, such compositions are prepared in
injectable formats either as liquid solutions or suspensions;
however, solid forms suitable for solution, or suspensions, in
liquid prior to use can also be prepared. The preparation can also
be emulsified. In particular, the pharmaceutical compositions may
be formulated in solid dosage form, for example capsules, tablets,
pills, powders, dragees or granules.
The choice of vehicle and the content of active substance in the
vehicle are generally determined in accordance with the solubility
and chemical properties of the active compound, the particular mode
of administration and the provisions to be observed in
pharmaceutical practice. For example, excipients such as lactose,
sodium citrate, calcium carbonate, dicalcium phosphate and
disintegrating agents such as starch, algenic acids and certain
complex silicates combined with lubricants such as magnesium
stearate, sodium lauryl sulphate and talc may be used for preparing
tablets. To prepare a capsule, it is advantageous to use lactose
and high molecular weight polyethylene glycols. When aqueous
suspensions are used they can contain emulsifying agents or agents
which facilitate suspension. Diluents such as sucrose, ethanol,
polyethylene glycol, propylene glycol, glycerol and chloroform or
mixtures thereof may also be used.
The lipophenolic compounds used according to the invention can be
administered in a suitable formulation to humans and animals by
topical or systemic administration, including oral, intrathecal,
enteral, parenteral (including subcutaneous, intra-arterial,
intramuscular, intravenous, intradermal), nutritional and
intraperitoneal.
In a particular embodiment, the lipophenolic compound according to
the invention is in a suitable formulation to humans, and in
particular is administered orally, intrathecally, enterally,
parenterally, nutritionally, and/or intraperitoneally.
In a preferred embodiment, the lipophenolic compound according to
the invention is in a suitable formulation for oral
administration.
In a particular embodiment, the lipophenolic compound according to
the invention will be in a suitable form selected from:
nano-suspension to improve their solubility and activity; natural
deep eutectic solvent (NADES), known as having high solubilizing
power, cheap components and low toxicity; NADES are composed of two
or more natural metabolites (amino acids, lipids, organic amines
and sugars) that when mixed together with certain ratio, forms a
liquid with lower melting point than any of their components (Dai
et al. 2013); in complex with protein to improve their solubility
and bioavailability (e.g.: albumin)
In a particular and preferred embodiment, the lipophenolic compound
formulated in a suitable form is a compound of formula (IIb)
##STR00058## with R.sub.3 selected from the group consisting
preferably of:
##STR00059## and more preferably
##STR00060## Additional Active Compounds
In a particular embodiment, the lipophenolic compound according to
the invention is associated with at least one additional active
compound selected from the group consisting of antibiotic,
antiviral, antifungal, antiparasitic, anti-inflammatory active
compound and mixtures thereof.
The man skilled in the art may refer to compounds usually known as
antibiotic, antiviral, antifungal, antiparasitic, anti-inflammatory
active compound and mixtures thereof.
The additional active compound may be administered simultaneously
or sequentially with the lipophenolic compound of the
invention.
In a first embodiment, the additional compound is present in the
same pharmaceutical composition than the lipophenolic compound.
In another embodiment, the additional compound is present in a
separate compartment or composition than the pharmaceutical
composition containing the lipophenolic compound.
In a particular embodiment, the lipophenolic compound formulated is
a compound of formula (IIb)
##STR00061## with R.sub.3 selected from the group consisting
preferably of:
##STR00062## and more preferably
##STR00063## associated with at least one additional active
compound selected from the group consisting of antibiotic,
antiviral, antifungal, antiparasitic, anti-inflammatory active
compound and mixtures thereof.
The man skilled in the art will choose the additional active
compound considering the disease or disorder to be prevented and/or
treated.
In particular, for a central nervous system disorder, he may use an
anti-inflammatory active compound.
In another embodiment, for viral hemorrhagic fever, he may use an
antiviral compound.
Short-Term Treatment
As new MMP-9 inhibitors, the lipophenolic compounds of the
invention will be used preferably not in a long-term continuous
treatment, but in a discontinuous or short-term intermittent
treatment administration.
By `discontinuous or intermittent` treatment according to the
invention, it means that the lipophenolic compounds are
administered for a short-term period, also named `short-term
treatment`, for example for 1 to 4 days, in particular for 1 day,
for 2 days, for 3 days or for 4 days, preferably for 1 day or 2
days.
In a particular embodiment, the lipophenolic compound according to
the invention may be administered before, during and/or after an
acute disorder phase (ex: acute inflammatory phase), for a
short-term treatment. The acute disorder phase may be forecasted,
in a subject susceptible to be affected, by specific markers
(including MMP-9) of the said disorder.
In another embodiment, the lipophenolic compound according to the
invention may be administered before, during and/or after a chronic
disorder phase, for a short-term treatment.
In a particular and preferred embodiment, the lipophenolic compound
according to the invention is administered for a short-term
treatment, for 1 to 4 days, and preferably just before or during
the acute phase with a potential or clear increased vascular
permeability.
The invention will be illustrated in the following non-limitative
figures and examples.
FIGURES
FIG. 1: Chemical and enzymatic synthesis of 4'-Resv-C.sub.22 or
4'-Resv-BE, 4'-Resv-DHA, 4'-Resv-LA and 4'-Resv-ALA.
FIG. 2: Chemical and enzymatic synthesis of 3-Resv-DHA.
FIG. 3: Assessment of anti-MMP-9 activity of resveratrol
lipophenolic derivatives.
FIG. 4: Zymogram and Curve of the dose-response effect of Resv-LA
(FIG. 4A) and Resv-C22 or Resv-BE (FIG. 4B) respectively on the
inhibitory MMP-9 activity.
FIG. 5: Assessment of in vitro vascular permeability assay of
resveratrol lipophenolic derivatives.
FIG. 6: Assessment of cell viability assay of Resv-LA (FIG. 6A) and
Resv-C22 or Resv-BE (FIG. 6B) respectively.
FIG. 7: Assessment of the inhibitory effect of Resv-LA (REV-LA) on
TNF-.alpha. release by LPS-activated microglia.
EXAMPLES
Materials and Methods
THP-1 cells (human monocytic THP-1 cell line) were cultured in 10%
heated inactivated FBS RPMI 1640 medium supplemented with
penicillin G 100 units/mL and streptomycin 100 .mu.g/mL purchased
from Fisher Scientific.TM. (Illkirch-Graffenstaden, France).
Human umbilical vascular endothelial cells (HUVECs) were cultured
in low serum (1%) EndoGro.TM. medium kit purchased from Merck
Millipore.TM. (Paris, France). TNF-.alpha. was purchased from
PeproTech.TM. (Neuilly-Sur-Seine, France).
Resveratrol has been isolated and purified from stalks of Vitis
vinifera, Vitaceae, according to the process described by (Delaunay
et al. 2002), SB-3CT #BML-E1325 (specific thiirane gelatinase
inhibitor used as a reference: it blocks laminin degradation by
MMP-9 so that prohibiting neuron apoptosis) was purchased from Enzo
Life Sciences.TM. (Villeurbanne, France) and dissolved in Dimethyl
sulfoxide (DMSO) Sigma-Aldrich.TM..
Human CD31/PECAM-1 antibody (BBA7) and Streptavidin-Fluorescein
(4800-30-14) were purchased from Bio-Techne.TM. (Abingdon, United
Kingdom), and all other chemicals used in this study are highly
purified molecular grade reagents.
Example 1: Synthesis of Resveratrol Derived Lipophenols
To evaluate the activity of different lipid chains at the 4'
position, Resv-C22 or Resv-BE (5a), Resv-LA (5b), Resv-DHA (5c) and
Resv-ALA (5d) were synthesized using enzymatic and chemical
synthesis starting from resveratrol (FIG. 1). In the first step,
the supported lipase Candida antartica (CALB, Novozyme 435,
selective of the 4' position) was used to introduce acetyl group
regio-selectively at the resveratrol C4OH position. The reaction
was performed in good yield (85%) without any acetyl derivatives at
the 3 or 5 positions. Hydroxyl groups at 3 and 5 positions of
compound 1 were then protected by triisopropylsilyl (TIPS)
protecting groups using triflate reagent (TIPS-OTf) and
diisopropylethylamine (DIPEA) as a base to obtain the protected
derivative 2. The acetyl group of compound 2 was deprotected with a
solution of sodium methanolate (MeONa) in anhydrous methanol and
resulted resveratrol-diTIPS (3) in an excellent yield of 95%. The
coupling reactions between compound 3 and the difference fatty
acid, docosanoic acid or behenic acid (C22), linoleic acid (LA),
docosahexaenoic acid (DHA) and linolenic acid (ALA) were initiated
using dicyclohexylcardodiimide and dimethylaminopyridine (DCC/DMAP)
as coupling reagents to access 4a-d. Final deprotection of TIPS
protecting groups by Et.sub.3N-3HF in dry tetrahydrofuran (THF)
yielded final lipophenols 5a-d.
In order to study the importance of the position of the lipidic
part on the resveratrol structure, the synthesis of a lipidic
resveratrol having the fatty acid at the 3 position was developed.
Starting from the fully protected resveratrol 2, one TIPS group at
the 5 position was removed using mild Et.sub.3N-3HF carefully
monitored by thin layer chromatography (48%, FIG. 2). Then, the
mono deprotected derivative 6 was linked to the fatty acid (DHA)
using DCC/DMAP. In order to preserve the ester linkage of the
compound 7, the acetate group was deprotected using enzymatic
lipase CALB in presence of butanol (89%) instead of MeONa solution.
The final TIPS deprotection using Et.sub.3N-3HF afforded the
desired 3-Resv-DHA compound (9).
Experimental Part
(E)-4-(3,5-dihydroxystyryl)phenyl acetate (Compound 1)
resveratrol (2.88 g, 12.61 mmol) was dissolved in
2-methylbutan-2-ol (280 mL) and vinyl acetate (72.40 mL, 756.70
mmol) in presence of the supported lipase Candida Antarctica
(Novozyme 435, CaIB, 14.40 g). The mixture was stirred with a
rotary evaporator at 40.degree. C. during 4 days, protected from
sunlight by aluminium foil. The lipase was then filtered off and
washed with AcOEt (10.times.50 mL) and diethyl ether (2.times.50
mL). The filtrate obtained was concentrated under reduced pressure
and the residue obtained was purified by chromatography on silica
gel using solid deposit (CH.sub.2Cl.sub.2/MeOH 99/1 to 98/2) to
give the 4'-O-acetyl resveratrol 1 (2.89 mg, 85%) as white solid.
R.sub.f (CH.sub.2Cl.sub.2/MeOH 95/5) 0.3; .sup.1H NMR (500 MHz;
CD.sub.3OD) .delta..sub.H 7.54 (d, J=7.6 Hz, 2H, H.sub.2' and
H.sub.6'), 7.07 (d, J=7.6 Hz, 2H, H.sub.3' and H.sub.5'), 7.04 (d,
J=16.2 Hz, 1H, H.sub.8), 6.97 (d, J=16.2 Hz, 1H, H.sub.7), 6.49 (s,
2H, H.sub.2, H.sub.6), 6.21-6.19 (m, 1H, Ha), 2.27 (s, 3H,
CH.sub.3(OAc)); .sup.13C NMR (125 MHz; MeOD) .delta..sub.C 171.1,
159.7 (2C), 151.5, 140.6, 136.6, 130.2, 128.3, 128.3, 122.9 (2C),
122.9 106.1 (2C), 103.2, 20.9.
(E)-4-(3,5-bis((triisopropylsilyl)oxy)styryl)phenyl acetate
(Compound 2)
4'-O-acetyl resveratrol 1 (3.18 g, 11.78 mmol) was dissolved in dry
THF (160 mL). DIPEA (4.20 mL, 24.70 mmol) and TIPS-OTf (6.70 mL,
24.70 mmol) were added dropwise to the solution and the reaction
mixture was stirred at room temperature during 4.5 h. Additional
amount of DIPEA (1.0 mL, 5.90 mmol) and TIPS-OTf (1.6 mL, 5.90
mmol) were added to reach completion of the reaction. After 2.5
additional hours of reaction, the solvent was evaporated under
reduced pressure. The residue obtained was dissolved in 200 mL of
AcOEt and washed with water ((2.times.100 mL) and brine (100 mL).
The organic phase was dried (MgSO.sub.4) and concentrated under
vacuum. The residue obtained was purified by chromatography on
silica gel (pentane/AcOEt 99/1 to 70/30) to give the protected
resveratrol 2 (6.85 g, 90%) as a colorless oil. R.sub.f
(pentane/AcOEt 95/5) 0.5; .sup.1H NMR (500 MHz; CDCl.sub.3)
.delta..sub.H 7.51 (d, J=8.0 Hz, 2H, H.sub.2' and H.sub.6'), 7.09
(d, J=8.0 Hz, 2H, H.sub.3' and H.sub.5'), 6.98 (d, J=16.3 Hz, 1H,
H.sub.8), 6.92 (d, J=16.3 Hz, 1H, H.sub.7), 6.65 (s, 2H, H.sub.2,
H.sub.6), 6.37-6.36 (m, 1H, Ha), 2.31 (s, 3H, CH.sub.3(OAc)), 1.26
(m, 6H, CH--Si), 1.12 (d, J=7.6 Hz, 36H, (CH.sub.3--CH); .sup.13C
NMR (125 MHz; CDCl.sub.3) .delta..sub.C 169.7, 157.3 (2C), 150.2,
139.0, 135.3, 129.3, 127.8, 127.7 (2C), 122.0 (2C), 111.6 (2C),
111.5, 21.4, 18.2 (6C), 12.9 (12C).
(E)-4-(3,5-bis((triisopropylsilyl)oxy)styryl)phenol (Compound
3)
The protected resveratrol 2 (6.85 g, 10.59 mmol) was dissolved in
dry MeOH (58 mL) and CH.sub.2Cl.sub.2 (28 mL). Sodium methoxide
(191 mg, 3.53 mmol) was added to the solution and the reaction
mixture was stirred at room temperature during 4.5 h. Further, 0.3
eq of NaOMe (191 mg, 3.53 mmol) was added to drive the reaction to
completion. After additional 2 h, the solvent was evaporated under
reduced pressure. The residue obtained was purified by
chromatography on silica gel (pentane/AcOEt 96/4 to 90/10) to give
the 4'-deprotected resveratrol 3 (6.04 g, 95%) as an colorless oil.
R.sub.f (hexane/AcOEt 90/10) 0.41; .sup.1H NMR (500 MHz;
CD.sub.3OD) .delta..sub.H 7.38 (d, J=8.5 Hz, 2H, H.sub.2' and
H.sub.6), 6.95 (d, J=16.2 Hz, 1H, H.sub.8), 6.84 (d, J=16.2 Hz, 1H,
H.sub.7), 6.77 (d, J=8.5 Hz, 2H, H.sub.3' and H.sub.5), 6.64-6.63
(m, 2H, H.sub.2, H.sub.6), 6.30-6.29 (m, 1H, H.sub.4), 1.30-1.22
(m, 6H, CH--Si), 1.14 (d, J=7.5 Hz, 36H, CH.sub.3--CH); .sup.13C
NMR (125 MHz; MeOD) .delta..sub.C 158.5, 158.3 (2C), 141.3, 130.1,
129.9, 129.0 (2C), 126.5, 116.5 (2C), 112.1 (2C), 111.4, 18.4 (6C),
13.9 (12C).
Description of 4'-Resv-C22 (4-Resv-BE) Series
(E)-4-(3,5-bis((triisopropylsilyl)oxy)styryl)phenyl docosanoate
(Compound 4a)
Compound 3 (3.00 g, 5.55 mmol) and docosanoic acid also named
behenic acid (2.27 g, 6.67 mmol) were partially dissolved in dry
DCM (180 mL) and the required amount of dry DMF (55 mL) was added
to solubilize the acid entirely. Afterwards, DCC (1.70 g, 8.33
mmol) and DMAP (339 mg, 2.78 mmol) were added and the reaction was
stirred at room temperature under argon until the conversion was
completed according to TLC. After 6 h, the reaction was stored into
the fridge (4.degree. C.) to allow the formation of DCU
precipitate, which was then filtered on frit. DCM (65 mL) was added
to the filtrate and it was washed with water (2.times.150 mL) and
brine (150 mL). The organic layer was dried over MgSO.sub.4 and
evaporated to gain 8.00 g of crude product. Purification was
performed by chromatography on silica gel (pentane/EtOAc 99:1) to
yield 4a (6.63 g, 76%) as a white solid.
R.sub.f (pentane/EtOAc 99:1) 0.4; .sup.1H NMR (500 MHz,
CDCl.sub.3): .delta..sub.H 7.50 (d, J=8.5 Hz, 2H, H.sub.2' and
H.sub.6'), 7.07 (d, J=8.5 Hz, 2H, H.sub.3' and H.sub.5), 6.99 (d,
J=16.0 Hz, 1H, H.sub.8), 6.92 (d, J=16.0 Hz, 1H, H.sub.7), 6.65 (d,
J=2.0 Hz, 2H, H.sub.2 and H.sub.6), 6.36 (t, J=2.5 Hz, 1H, Ha),
2.56 (t, J=7.5 Hz, 2H, CH.sub.2--C.dbd.O), 1.76 (quint, J=7.5 Hz,
2H, CH.sub.2--CH.sub.2--C.dbd.O), 1.43-1.22 (m, 42H,
CH.sub.2--CH.sub.2 and CH--Si), 1.11 (d, J=7.5 Hz, 36H,
CH.sub.3--CH), 0.89 (t, J=7.1 Hz; 3H, CH.sub.3--CH.sub.2). .sup.13C
NMR (126 MHz, CDCl.sub.3) .delta..sub.C 172.4, 157.2, 157.2, 150.2,
138.9, 135.1, 129.1, 127.7, 127.5 (2C), 121.9 (2C), 111.5 (2C),
111.4, 34.6, 32.1, 29.9 (8C), 29.8, 29.8, 29.8, 29.7, 29.6, 29.5,
29.4, 29.2, 25.1, 22.8, 18.1 (6C), 14.3, 12.8 (12C).
(E)-4-(3,5-dihydroxystyryl)phenyl docosanoate (Compound 5a)
Compound 4a (3.63 g, 4.20 mmol) was dissolved in dry THF (220 mL)
under argon atmosphere. Addition of Et.sub.3N-3HF (4.11 mL, 25.21
mmol) was arranged via plastic syringe and the reaction was allowed
to stir at room temperature and monitored by TLC (pentane/EtOAc 7:3
and 9:1). Additional equivalents of Et.sub.3N-3HF were added after
3.5 h (2.06 mL, 12.61 mmol) and 6 h (2.06 mL, 12.61 mmol) of
reaction. After 8 hours, the THF was evaporated under reduced
pressure and the residue was dissolved in 400 mL of EtOAc, and then
washed with H.sub.2O (3.times.200 mL) and brine (200 mL). The
organic layer was dried over MgSO.sub.4 and evaporated to gain 2.70
g of crude product. Purification was performed by chromatography on
silica gel using solid deposit (pentane/EtOAc 7:3 to 0:1) and
resulted in 503 mg (17%) of mono protected derivative and 1.82 g
(79%) of compound 5a as a white solid.
R.sub.f (pentane EtOAc 7:3) 0.3; .sup.1H NMR (500 MHz,
CDCl.sub.3/MeOD 10:1) .delta..sub.H 7.31 (d, J=8.5 Hz, 2H, H.sub.2'
and H.sub.6'), 6.87 (d, J=8.5 Hz, 2H, H.sub.3' and H.sub.5'), 6.84
(d, J=16.5 Hz, 1H, H.sub.8), 6.74 (d, J=16.5 Hz, 1H, H.sub.7), 6.34
(d, J=2.0 Hz, 2H, H.sub.2 and H.sub.6), 6.08 (t, J=2.0 Hz, 1H,
H.sub.4), 2.39 (t, J=7.0 Hz, 2H, CH.sub.2--C.dbd.O), 1.57 (quint,
J=7.0 Hz, 2H, CH.sub.2--CH.sub.2--C.dbd.O), 1.23-1.07 (m, 36H,
CH.sub.2--CH.sub.2), 0.69 (t, J=6.6 Hz, 3H, CH.sub.3--CH.sub.2);
.sup.13C NMR (126 MHz, CDCl.sub.3/MeOD 10:1) .delta..sub.C 172.8,
157.9 (2C), 149.8, 139.1, 135.0, 128.9, 127.3, 127.2 (2C), 121.6
(2C), 105.1 (2C), 102.1, 34.2, 31.7, 29.5 (9C), 29.4, 29.4, 29.4,
29.3, 29.2, 29.0, 28.9, 24.7, 22.5, 13.8.
Description of 4'-Resv-LA Series
(9,12Z)-4-((E)-3,5-bis((triisopropylsilyl)oxy)styryl)phenyl
octadeca-9,12-dienoate (Compound 4b)
Compound 3 (1.00 g, 1.67 mmol) and linoleic acid (LA; 623 mg, 2.22
mmol) were dissolved in dry DCM (40 mL). Next, DCC (573 mg, 2.78
mmol) and DMAP (113 mg, 0.93 mmol) were added to the reaction
mixture which was stir at room temperature under inert atmosphere
(monitored by TLC pentane/EtOAc 95:5). The reaction was terminated
after 3 hours. Flask was put into the fridge (4.degree. C.) for 1 h
min to maximize the amount of DCU crystals. White DCU precipitate
was then removed by filtration on frit, rinsed by a few drops of
cold DCM. Filtrate was diluted by 40 mL of DCM and washed twice
with water (30 mL) and once with brine (30 mL). Aqueous phases were
re-extracted with 100 mL of DCM. Organic layers were collected,
dried over MgSO.sub.4 and evaporated. Purification by silica gel
column chromatography (pentane/EtOAc 99.5/5 to 99/1) resulted in
1.10 g (74%) of compound 4b (colorless oil).
Rf (pentane/EtOAc 95:5) 0.7; .sup.1H NMR (500 MHz, CDCl.sub.3):
.delta..sub.H 7.79 (d, J=8.5 Hz, 2H, H.sub.2' and H.sub.6'), 7.06
(d, J=8.5 Hz, 2H, H.sub.3' and H.sub.5'), 6.97 (d, J=16 Hz, 1H,
H.sub.8), 6.91 (d, J=16 Hz, 1H, H.sub.7), 6.64 (d, J=1.5 Hz, 2H,
H.sub.2 and H.sub.6), 6.3 (t, J=1.5 Hz, 1H, H.sub.4), 5.41-5.33 (m,
4H, CH.dbd.CH), 2.78 (t, J=6.5 Hz, 2H, CH.sub.2 bis-allylic), 2.55
(t, J=7.5 Hz, 2H, CH.sub.2--C.dbd.O), 2.07-2.03 (m, 4H, CH.sub.2
allylic), 1.75 (quint, J=7.5 Hz, 2H, CH.sub.2--CH.sub.2--C.dbd.O),
1.42-1.22 (m, 20H, CH.sub.2--CH.sub.2 and CH--Si), 1.11 (d, J=7.5
Hz, 36H, CH.sub.3--CH), 0.89 (t, J=7 Hz, 3H, CH.sub.3--CH.sub.2);
.sup.13C NMR (126 MHz, CDCl.sub.3) .delta..sub.C 172.6, 157.4 (2C),
150.4, 139.1, 135.3, 130.6, 130.3, 129.3, 128.4, 128.2, 127.9,
127.7 (2C), 122.1 (2C), 111.7 (2C), 111.6, 34.8, 31.8, 29.9, 29.7,
29.5, 29.4, 29.4, 27.5, 27.5, 26.0, 25.3, 22.9, 18.2 (6C), 14.4,
13.0 (12C).
(9,12Z)-4-((E)-3,5-dihydroxystyryl)phenyl octadeca-9,12-dienoate
(Compound 5b)
Et.sub.3N-3HF (1.32 mL, 8.08 mmol) was added via plastic syringe to
a solution of Compound 4b (1.08 g, 1.35 mmol) dissolved in dry THF
(60 mL) The reaction was stirred at room temperature under argon.
Further equivalents of Et.sub.3N-3HF (2.times.0.66 mL, 2.times.4.04
mmol) were added at four and six hours of reaction time. Reaction
was terminated after another two hours (reaction time 8 h,
monitored by TLC pentane/EtOAc 7:3 and 9:1). Reaction media was
evaporated and the residue was dissolved in EtOAc (120 mL). Organic
phases was washed with H.sub.2O (3.times.60 mL) and brine (60 mL),
dried over MgSO.sub.4, filtered and evaporated under reduced
pressure. Crude product was purified by column chromatography on
silica gel (pentane/EtOAc 7:3 to 6:4) to obtain 5b (546 mg, 83%) as
a white solid.
R.sub.f (pentane/EtOAc 7:3) 0.3; .sup.1H NMR (500 MHz, CDCl.sub.3)
.delta..sub.H 7.34 (d, J=8.5 Hz, 2H, H.sub.2' and H.sub.6'), 7.00
(d, J=8.5 Hz, 2H, H.sub.3' and H.sub.5'), 6.79 (d, J=16.5 Hz, 1H,
H.sub.8), 6.70 (d, J=16.5 Hz, 1H, H.sub.7), 6.40 (d, J=2.0 Hz, 2H,
H.sub.2 and H.sub.6), 6.23 (t, J=2.0 Hz, 1H, H.sub.4), 5.99 (Br,
2H, OH), 5.41-5.32 (m, 4H, CH.dbd.CH), 2.77 (t, J=6.5 Hz, 2H,
CH.sub.2 bis-allylic), 2.56 (t, J=7.5 Hz, 2H, CH.sub.2--C.dbd.O),
2.06-2.03 (m, 4H, CH.sub.2 allylic), 1.75 (quint, J=7.5 Hz, 2H,
CH.sub.2--CH.sub.2--C.dbd.O), 1.42-1.25 (m, 14H,
CH.sub.2--CH.sub.2), 0.88 (t, J=7 Hz, 3H, CH.sub.3--CH.sub.2);
.sup.13C NMR (126 MHz, CDCl.sub.3) .delta..sub.C 173.5, 157.0 (2C),
150.1, 139.7, 135.1, 130.4, 130.2, 128.4, 128.2, 128.2, 128.0,
127.7 (2C), 121.8 (2C), 106.3 (2C), 102.6, 34.6, 31.7, 29.7, 29.5,
29.3, 29.3, 29.2, 27.3, 27.3, 25.7, 25.0, 22.7, 14.2.
Description of 4'-Resv-DHA Series (4c and 5c)
The synthesis of Resv-DHA is described in the publication Crauste
et al. (2014).
(4,7,10,13,16,19
Z)-4-((E)-3,5-bis(triisopropylsilyloxy)styryl)phenyl
docosa-4,7,10,13,16,19-hexaenoate (Compound 4c)
Coupling of the di-protected resveratrol 3 (103 mg, 0.18 mmol) and
DHA (67 mg, 0.20 mmol) was performed with the general procedure and
afforded 4c (130 mg, 80%) as an uncolored oil after purification on
silicagel chromatography (hexane/AcOEt 99/1).
R.sub.f (hexane/AcOEt 95/5) 0.73; .sup.1H NMR (500 MHz; CDCl.sub.3)
.delta..sub.H 7.50 (d, J=8.5 Hz, 2H, H.sub.2' and H.sub.6'), 7.07
(d, J=8.4 Hz, 2H, H.sub.3' and H.sub.5), 6.98 (d, J=16.5 Hz, 1H,
H.sub.8), 6.92 (d, J=16.5 Hz, 1H, H.sub.7), 6.64 (d, J=2.3 Hz, 2H,
H.sub.2, H.sub.6), 6.36 (t, J=2.3 Hz 1H, H.sub.4), 5.50-5.29 (m,
12H, CH.dbd.CH), 2.90-2.80 (m, 10H, CH.sub.2 bis-allylic), 2.64 (t,
J=7.0 Hz, 2H, CH.sub.2--C.dbd.O), 2.55-2.51 (m, 2H, CH.sub.2
allylic), 2.08 (quint, J=7.0 Hz, 2H, CH.sub.2 allylic), 1.29-1.22
(m, 6H, CH--Si), 1.12 (d, J=7.5 Hz, 36H, (CH.sub.3).sub.2C); 0.98
(t, J=7.5 Hz, 3H, CH.sub.3); .sup.13C NMR (125 MHz; CDCl.sub.3)
.delta..sub.C 171.8, 157.4, 150.4, 139.1, 135.3, 132.3, 130.0,
129.3, 128.9, 128.7, 128.6, 128.6, 128.4, 128.4, 128.3, 128.2,
127.9, 127.8, 127.7, 127.3, 122.0, 111.7, 111.6, 34.6, 25.9, 25.9,
25.8, 23.1, 20.9, 18.2, 14.6, 13.0
(4,7,10,13,16,19 Z)-4-((E)-3,5-dihydroxyphenylstyryl)phenyl
docosa-4,7,10,13,16,19-hexaenoate (Compound 5c)
deprotection of the protected DHA-resveratrol 4c (142 mg, 0.17
mmol) was performed with the general procedure and afforded 5c (55
mg, 61%) as white solid after 7 h of reaction and purification on
silicagel chromatography (hexane/AcOEt 95/5 to 70/30).
R.sub.f (hexane/AcOEt 70/30)=0.22; .sup.1H NMR (500 MHz;
CDCl.sub.3) .delta..sub.H 7.45 (d, J=8.6 Hz, 2H, H.sub.2' and
H.sub.6'), 7.07 (d, J=8.5 Hz, 2H, H.sub.3' and HO, 6.95 (d, J=16.2
Hz, 1H, H.sub.8), 6.85 (d, J=16.2 Hz, 1H, H.sub.7), 6.51 (d, J=2.1
Hz, 2H, H.sub.2, H.sub.6), 6.26 (t, J=2.1 Hz, 1H, H.sub.4),
5.52-5.29 (m, 12H, CH.sub.2 bis-allylic), 5.13 (br, 2H, OH),
2.90-2.80 (m, 10H, CH.sub.2 allylic), 2.66 (t, J=7.4 Hz, 2H,
CH.sub.2--C.dbd.O), 2.52-2.56 (m, 2H, CH.sub.2 allylic), 2.08
(quint, J=7.8 Hz, 2H, CH.sub.2 allylic), 0.98 (t, J=7.3 Hz, 3H,
CH.sub.3); .sup.13C NMR (125 MHz; CDCl.sub.3) .delta..sub.C 172.3,
157.3, 150.4, 140.0, 135.2, 132.4, 130.1, 128.9, 128.7, 128.6,
128.6, 128.6, 128.5, 128.4, 128.4, 128.3, 128.2, 127.8, 127.7,
127.3, 122.1, 106.4, 102.7, 34.6, 25.9, 25.8, 23.1, 20.9, 14.6
Description 4'-Resv-ALA Series
(9,12, 15Z)-4-((E)-3,5-bis((triisopropylsilyl)oxy)styryl)phenyl
octadeca-9,12,15-trienoate (Compound 4d)
A solution of linolenic acid (ALA; 56 mg, 0.20 mmol) in dry DCM
(2.50 mL) was added to the protected resveratrol 3 (100 mg, 0.18
mmol) in solution in DCM (2.50 mL). DCC (42 mg, 0.20 mmol) and DMAP
(6 mg, 0.05 mmol) were added to the reaction mixture and the
solution was left to stir at room temperature under inert
atmosphere during 2 h (monitored by TLC pentane/EtOAc 95:5). Flask
was put into the fridge (4.degree. C.) for 1 h to maximize the
amount of DCU crystals. White DCU precipitate was then removed by
filtration on frit, rinsed by a few drops of cold DCM. Filtrate was
diluted by 10 mL of DCM and washed twice with water (10 mL) and
once with brine (10 mL). The organic layer was dried over
MgSO.sub.4 and evaporated. Purification by silica gel column
chromatography (pentane/EtOAc 99.5/5 to 99/1) resulted in 115 mg
(76%) of compound 4d as a colorless oil.
Rf (pentane/EtOAc 95:5) 0.5; .sup.1H NMR (500 MHz, CDCl.sub.3):
.delta..sub.H 7.50 (d, J=8.5 Hz, 2H, H.sub.2' and HO, 7.07 (d,
J=8.5 Hz, 2H, H.sub.3' and HO, 6.98 (d, J=16.5 Hz, 1H, H.sub.8),
6.92 (d, J=16.5 Hz, 1H, H.sub.7), 6.65 (d, J=2.5 Hz, 2H, H.sub.2
and H.sub.6), 6.36 (t, J=2.5 Hz, 1H, Ha), 5.39-5.36 (m, 6H,
CH.dbd.CH), 2.82 (t, J=6.5 Hz, 4H, CH.sub.2 bis-allylic), 2.56 (t,
J=7.5 Hz, 2H, CH.sub.2--C.dbd.O), 2.10-2.06 (m, 4H, CH.sub.2
allylic), 1.75 (quint, J=7.5 Hz, 2H, CH.sub.2--CH.sub.2--C.dbd.O),
1.43-1.21 (m, 14H, CH.sub.2--CH.sub.2 and CH--Si), 1.11 (d, J=7.5
Hz, 36H, CH.sub.3--CH), 0.89 (t, J=7 Hz, 3H, CH.sub.3--CH.sub.2);
.sup.13C NMR (126 MHz, CDCl.sub.3) .delta..sub.C 172.3, 157.2,
150.2, 138.9, 135.1, 132.1, 132.1, 130.4, 129.1, 128.4, 128.4,
127.9, 127.7, 127.5 (2C), 127.2, 121.9 (2C), 111.5 (2C), 111.4,
34.5, 29.7, 29.3, 29.2, 29.2, 27.3, 25.7, 25.6, 25.0, 20.7, 18.1
(6C), 14.4, 12.8 (12C).
(9, 12, 15Z)-4-((E)-3,5-dihydroxystyryl)phenyl
octadeca-9,12-dienoate (Compound 5d)
Et.sub.3N-3HF (134 .mu.l, 0.82 mmol) was added via plastic syringe
to a solution of ALA-resveratrol 4d (110 mg, 0.14 mmol) dissolved
in dry THF (6 mL). Further equivalents of Et.sub.3N-3HF (2.times.70
.mu.L, 2.times.0.41 mmol) were added at four and six hours of
reaction time. Reaction was terminated after another two hours
(reaction time 8 h, monitored by TLC pentane/EtOAc 7:3 and 9:1).
Reaction media was evaporated and the residue was dissolved in
EtOAc (20 mL) and then washed with H.sub.2O (3.times.20 mL) and
brine (20 mL). Organic phase was dried over MgSO.sub.4, filtered
and evaporated under reduced pressure. Crude product was purified
by column chromatography on silica gel (cyclohexane/AcOEt 80/20)
and afforded Resv-ALA 5d (57 mg, 84%) as white solid.
Rf (pentane/EtOAc 7/3) 0.3; .sup.1H NMR (500 MHz, CDCl.sub.3):
.delta..sub.H 7.38 (d, J=8.5 Hz, 2H, H.sub.2' and HO, 7.02 (d,
J=8.5 Hz, 2H, H.sub.3' and HO, 6.84 (d, J=16.5 Hz, 1H, H.sub.8),
6.75 (d, J=16.5 Hz, 1H, H.sub.7), 6.43 (d, J=2.0 Hz, 2H, H.sub.2
and H.sub.6), 6.23 (t, J=2.0 Hz, 1H, Ha), 5.41-5.32 (m, 6H,
CH.dbd.CH), 2.81 (t, J=6.0 Hz, 4H, CH.sub.2 bis-allylic), 2.57 (t,
J=7.5 Hz, 2H, CH.sub.2--C.dbd.O), 2.11-2.04 (m, 4H, CH.sub.2
allylic), 1.75 (quint, J=7.5 Hz, 2H, CH.sub.2--CH.sub.2--C.dbd.O),
1.42-1.30 (m, 8H, CH.sub.2--CH.sub.2), 0.97 (t, J=7.5 Hz, 3H,
CH.sub.3--CH.sub.2); .sup.13C NMR (126 MHz, CDCl.sub.3)
.delta..sub.C 173.3, 157.0 (2C), 150.1, 139.7, 135.0, 132.1, 130.4,
128.4, 128.4, 128.4, 128.2, 127.9 (2C), 127.7, 127.2, 121.9 (2C),
106.3 (2C), 102.6, 34.6, 29.7, 29.3, 29.2, 26.2, 27.3, 25.7, 25.6,
25.0, 20.7, 14.4.
Description 3-Resv-DHA Series
(E)-4-(3-hydroxy-5-((triisopropylsilyl)oxy)styryl)phenyl acetate
(Compound 6)
Et.sub.3N-3HF (554 .mu.L, 3.40 mmol) was added dropwise via plastic
syringe to a solution protected resveratrol 2 (1.00 g, 1.70 mmol)
dissolved in dry THF (60 mL). The reaction was stirred at room
temperature during 3 h. AcOEt (60 mL) was added to the mixture and
the organic layer was washed with water (20 mL) and brine (20 mL).
The organic phase was dried on MgSO.sub.4 and concentrated under
reduced pressure. The residue obtained was purified by
chromatography on silica gel (cyclohexane/AcOEt 95/5 to 80/20) to
give the mono-protected resveratrol 6 (350 mg, 48%) as a white
solid. The di-deprotected resveratrol was isolated in 26% as a
white solid (118 mg).
R.sub.f (Hexane/AcOEt 70/30) 0.6; 1H NMR (500 MHz, CDCl.sub.3)
.delta..sub.H 7.46 (d, J=8.5 Hz, 2H, H.sub.2' and H.sub.6'), 7.07
(d, J=8.5 Hz, 2H, H.sub.3' and H.sub.5'), 6.93 (d, J=16.5 Hz, 1H,
H.sub.8), 6.86 (d, J=16.5 Hz, 1H, H.sub.7), 6.59 (t, J=1.5 Hz, 1H,
H.sub.2), 6.51 (s, 1H, H.sub.4), 6.32 (t, J=2.0 Hz, 1H, H.sub.6),
5.55 (Br, 1H, OH), 2.32 (s, 3H, CH.sub.3--CO), 1.32-1.23 (m, 3H,
CH--Si), 1.12 (d, J=7.0 Hz, 18H, CH.sub.3--CH); .sup.13C NMR (126
MHz, CDCl.sub.3) .delta..sub.C 170.2, 157.5, 156.9, 150.1, 139.2,
135.2, 128.9, 127.9, 127.6 (2C), 121.8 (2C), 111.3, 107.0, 106.3,
21.3, 18.1 (3C), 12.8 (6C).
(4,7,10,13,16,19Z)-3-((E)-4-acetoxystyryl)-5-((triisopropylsilyl)oxy)pheny-
l docosa-4,7,10,13,16,19-hexaenoate (Compound 7)
Compound 6 (470 mg, 1.1 mmol) and DHA (397 mg, 1.2 mmol) were
dissolved in dry DCM (20 mL) under argon. Then, DCC (250 mg, 1.21
mmol) and DMAP (13 mg, 0.1 mmol) were added to the reaction mixture
and the solution was stirred at room temperature under inert
atmosphere during 2 h (monitored by TLC pentane/EtOAc 90:10). The
reaction was put into the fridge (4.degree. C.) for 1 h to maximize
the amount of DCU crystals. White DCU precipitate was then removed
by filtration on frit, rinsed by a few drops of cold DCM. Filtrate
was diluted by 20 mL of DCM and washed twice with water (15 mL) and
once with brine (15 mL). Aqueous phase was re-extracted with 50 mL
of DCM. Organic layers were collected, dried over MgSO.sub.4 and
evaporated. Purification on silica gel chromatography
(cyclohexane/AcOEt 98/2) afforded compound 7 (391 mg, 49%) as a
white solid.
R.sub.f (Hexane/AcOEt 90/10) 0.5; .sup.1H NMR (500 MHz, CDCl.sub.3)
.delta..sub.H 7.49 (d, J=8.7 Hz, 2H, H.sub.2' and H.sub.6), 7.08
(d, J=8.7 Hz, 2H, H.sub.3' and H.sub.5'), 7.00 (d, J=16.2 Hz, 1H,
H.sub.8), 6.93 (d, J=16.2 Hz, 1H, H.sub.7), 6.84 (t, J=2.0 Hz, 2H,
H.sub.2 and H.sub.6), 6.53 (t, J=2.0 Hz, 1H, H.sub.4), 5.50-5.28
(m, 12H, CH.dbd.CH), 2.89-2.79 (m, 10H, CH.sub.2 bis-allylic),
2.63-2.60 (m, 2H, CH.sub.2--C.dbd.O), 2.56-2.50 (m, 2H, CH.sub.2
allylic), 2.30 (s, 3H, CH.sub.3--C.dbd.O), 2.10-2.04 (m, 2H,
CH.sub.2 allylic), 1.30-1.23 (m, 3H, CH--Si), 1.12 (d, J=7.3, 18H,
CH.sub.3--CH), 0.97 (t, J=7.5, 3H, CH.sub.3--CH.sub.2); .sup.13C
NMR (126 MHz, CDCl.sub.3) .delta..sub.C 171.4, 169.5, 157.1, 151.8,
150.3, 139.2, 134.9, 132.1, 129.8, 128.7, 128.7, 128.5, 128.4,
128.4, 128.3, 128.2, 128.2, 128.1, 128.0, 127.7, 127.6 (2C), 127.1,
121.9 (2C), 115.8, 112.9, 112.3, 34.4, 25.8 (2C), 25.7, 25.7, 25.6,
22.9, 21.3, 20.7, 18.0 (3C), 14.4, 12.7 (6C).
(4,7,10,13,16,19Z)-3-((E)-4-hydroxystyryl)-5-((triisopropylsilyl)oxy)pheny-
l docosa-4,7,10,13,16,19-hexaenoate (Compound 8)
The protected DHA-resveratrol 7 (345 mg, 0.47 mmol) was dissolved
in t-buthylmethylether (55 mL) and n-BuOH (2 mL). The supported
lipase Candida Antarctica (Novozyme 435, CaIB, 345 mg) was added to
the solution and the mixture was stirred at 40.degree. C. during 3
days. The lipase was filtered off and washed with 5.times.30 mL of
AcOEt and 2.times.30 mL of diethyl ether. The filtrate was
concentrated under reduced pressure and the residue obtained was
purified by chromatography on silica gel (cyclohexane/AcOEt 95/5 to
90/10) to give the compound 8 (291 mg, 89%) as a yellow oil.
R.sub.f (Hexane/AcOEt 90/10) 0.55; .sup.1H NMR (500 MHz,
CDCl.sub.3) .delta..sub.H 7.37 (d, J=8.6 Hz, 2H, H.sub.2' and
H.sub.6'), 6.96 (d, J=16.2 Hz, 1H, H.sub.8), 6.85-6.79 (m, 5H,
H.sub.3', H.sub.5', H.sub.2, H.sub.6 and H.sub.7), 6.50 (t, J=2.1
Hz, 1H, H.sub.4), 5.48-5.28 (m, 12H, CH.dbd.CH), 2.89-2.79 (m, 10H,
CH.sub.2 bis-allylic), 2.63-2.60 (m, 2H, CH.sub.2--C.dbd.O),
2.56-2.50 (m, 2H, CH.sub.2 allylic), 2.10-2.04 (m, 2H, CH.sub.2
allylic), 1.30-1.24 (m, 3H, CH--Si), 1.11 (d, J=7.4 Hz, 18H,
CH.sub.3--CH), 0.97 (t, J=7.5 Hz, 3H, CH.sub.3--CH.sub.2); .sup.13C
NMR (126 MHz, CDCl.sub.3) .delta..sub.C 171.5, 157.0, 155.5, 151.8,
139.7, 132.2, 130.1, 129.8, 129.2, 128.7, 128.5, 128.4, 128.4,
128.2, 128.2, 128.2 (2C), 128.1, 128.0, 127.7, 127.2, 126.0, 115.7
(2C), 115.5, 112.5, 112.1, 34.5, 25.8 (2C), 25.8, 25.8, 25.7, 22.9,
20.7, 18.1 (3C), 14.4, 12.8 (6C).
(4,7,10,13,16,19Z)-3-hydroxy-5-((E)-4-hydroxystyryl)phenyl
docosa-4,7,10,13,16,19-hexaenoate (Compound 9)
Et.sub.3N-3HF (232 .mu.l, 1.41 mmol) was added via plastic syringe
to a solution of DHA-resveratrol 8 (330 mg, 0.47 mmol) dissolved in
dry THF (20 mL) The reaction was stirred at room temperature under
argon during 4 h30. TLC monitoring was carried out using
pentane/EtOAc (7:3 and 9:1) to observe the desired product
formation and the departure of starting material respectively.
Reaction media was evaporated and the residue was dissolved in
EtOAc (20 mL) and then washed with H.sub.2O (20 mL) and brine (20
mL). Organic phase was dried over MgSO.sub.4, filtered and
evaporated. Crude product was purified by column chromatography on
silica gel (Cyclohexane/AcOEt 80/20) and afforded 3-Resv-DHA 9 (191
mg, 75%) as a white solid
R.sub.f (Hexane/AcOEt 70/30) 0.33; .sup.1H NMR (500 MHz,
CDCl.sub.3) .delta..sub.H 7.34 (d, J=8.6, 2H, H.sub.2' and
H.sub.6), 6.95 (d, J=16.2, 1H, H.sub.8), 6.83-6.73 (m, 5H,
H.sub.3', H.sub.5', H.sub.2, H.sub.6 and H.sub.7), 6.46 (t, J=2.1,
1H, H.sub.4), 5.51-5.28 (m, 12H, CH.dbd.CH), 5.13 (br, 1H, OH),
5.09 (br, 1H, OH), 2.89-2.79 (m, 10H, CH.sub.2 bis-allylic),
2.65-2.62 (m, 2H, CH.sub.2--C.dbd.O), 2.58-2.51 (m, 2H, CH.sub.2
allylic), 2.10-2.03 (m, 2H, CH.sub.2 allylic), 0.97 (t, J=7.5, 3H,
CH.sub.3--CH.sub.2); .sup.13C NMR (126 MHz, CDCl.sub.3)
.delta..sub.H 172.0, 156.6, 155.6, 151.8, 140.3, 132.2, 129.9,
129.9, 129.6, 128.7, 128.5, 128.4, 128.4, 128.2 (2C), 128.2, 128.2,
128.1, 128.0, 127.5, 127.1, 125.5, 115.8 (2C), 111.9, 110.9, 108.1,
34.5, 25.8 (2C), 25.8, 25.7, 25.6, 22.9, 20.7, 14.4.
Example 2: Effect of Lipophenolic Derivative of Resveratrol on
MMP-9 Activity
THP-1 cells were seeded (3.times.10.sup.5 cell/well) in 24-well
plate with 10 ng/mL TNF-.alpha. with or without 30 .mu.M of
resveratrol derivatives dissolved in DMSO. After 24 hours
incubation at 37.degree. C., supernatant was collected and tested
on zymography. Gel bands demonstrates MMP-9 activity of activated
THP-1 cell line in presence of resveratrol lipophenolic
derivatives.
Assessment of MMP-9 Activity in THP-1 Cell Line
Low-serum (1%) RPMI-1640 medium was used for the assessment of
MMP-9 activity on TNF-alpha activated THP-1 cell line
3.times.10.sup.6 cell/mL. Firstly, resveratrol and its derived
lipophenolic compounds were dissolve in DMSO, and incubated at a
final concentration of 30 .mu.M with 10 ng/mL TNF-alpha treated
THP-1 in CO.sub.2 incubator chamber for 24 hours at 37.degree. C.
After incubation, cell suspension was centrifuged at 1200 r.p.m for
five minutes, supernatant was recovered and stored at -80.degree.
C. for zymogram analysis.
Zymogram
The anti MMP-9 activity was assessed using gelatin zymography. In
brief, the collected supernatants were loaded on 10%
SDS-polyacrylamide gel electrophoresis (PAGE) supplemented with 1%
gelatin without reducing agents. After separation, gels were washed
three times with 2.5% Triton X-100 and incubated with gelatinase
buffer (NaCl 200 mM, Tris Base 50 mM, CaCl2) 5 mM and ZnCl2 0.25
mM; pH 7.5), for 24 hr at 37.degree. C. on an orbital shaker at 100
r.p.m/min. Gels were further stained for one hour with Commassie
Blue-staining solution (0.025% Commassie Blue, 40% methanol and 10%
acetic acid) followed by destaining with 20% methanol and 10%
glacial acetic acid solution until the clear bands appearance. Gels
were photographed and analyzed by GelAnalyzer 2010a.TM.
software.
The results are presented in FIG. 3, showing that Resv-LA, Resv-C22
(Resv-BE), and to a lesser extent 4'-Resv-DHA, demonstrated
inhibition of MMP9-activity in TNF-alpha-activated THP-1 monocytes
(FIG. 3). While 4'-Resv-DHA reduced MMP-9 activity at 30 .mu.M, its
regio-isomer, having the lipid chain linked with the hydroxyl group
in position 3 of resveratrol, the 3-Resv-DHA (FIG. 2), was not
active at the same concentration.
Resv-LA and Resv-C22 (Resv-BE) have demonstrated interesting
inhibition of activity of MMP-9 (FIG. 3).
And the results in FIG. 4 showed a dose-response effect of Resv-LA
(FIG. 4A) and respectively Resv-C22 (Resv-BE) (FIG. 4B) on the
inhibitory activity of MMP9.
Example 3: Effect of Lipophenolic Derivative of Resveratrol on In
Vitro Vascular Permeability Assay (FITC-Dextran Endothelial
Permeability Assay)
The permeability of HUVEC monolayer seeded on collagen coated
semi-permeable inserts was examined using in vitro vascular
permeability assay kit, Millipore.TM. (Paris, France) according to
the manufacturer protocol.
Endothelial cells are seeded into the inserts and cultured until
complete monolayer formation occurs. After forming confluent
monolayer by seeding HUVECs (4.times.10.sup.5 cells/insert) for 48
hours, medium was replaced with 100 ng/mL TNF-.alpha. in medium
with and without MMP-9 inhibitors at 10 .mu.M and incubated for 24
hours in CO.sub.2 chamber at 37.degree. C. Lipophenolic derivatives
of resveratrol were dissolved in DMSO, and SB-3CT (gelatinase
inhibitor) was dissolved in DMSO.
At the end of the permeability treatment, 150 .mu.l FITC-dextran in
media solution were incubated for 20 minutes at room temperature
protected from light. FITC-dextran permeates the treated cell
monolayer into the plate well. Permeation was stopped by removing
the inserts and 100 .mu.l were withdrawn from the receiving tray
and added to 96-well opaque plate for fluorescence measurement. The
resulting fluorescence in the plate well is measured and used as an
indicator of the extent of monolayer permeability. Filters used are
485 nm and 535 nm for excitation and emission, respectively.
The results are presented in FIG. 5, showing that Resv-LA
demonstrated inhibition of TNF-alpha-enhanced permeability of the
HUVEC monolayer.
Example 4: Effect of Lipophenolic Derivative of Resveratrol on Cell
Viability Assay (Mtt Assay)
The MTT assay is a test used to evaluate the cytotoxicity of
compounds.
The cytotoxicity assay was carried out as described by Mosmann
(Mosmann 1983). HUVEC cells and THP-1 cell line (1.times.10.sup.4
cell/well) were seeded in 96-well plate and incubated CO.sub.2
incubator chamber at 37.degree. C. for 24 h. Cells were treated
with serial dilutions of lipophenolic derivative of resveratrol at
a final concentration of 10, 20, 40 and 80 .mu.M, and plate was
incubated for 72 hours. Supernatant was discarded and
MTT-serum-free medium was added to each plate and incubated for
three additional hours. The formed formazan blue crystals were
further dissolved by addition of 100 .mu.L of 10% SDS in 0.1 N HCl
to each plate for 2 hours. The optical density was measured at 570
nm (reference filter 690 nm) using a TECAN.TM. plate reader. Lethal
Concentration 50% (LC50) was calculated using GraphPad Prism v.5
software.
The results are presented in FIG. 6, showing that Resv-LA and
Resv-C22 (Resv-BE) at concentration 10, 20, 40 .mu.M does not have
cytotoxic effect on viability.
All these results demonstrate that the lipophenolic compounds of
the invention, in particular Resv-LA (FIG. 6A) and Resv-C22
(Resv-BE) (FIG. 6B), have inhibition effect of activity of MMP-9
and are able to decrease the TNF-alpha induced endothelial
permeability. Such lipophenolic compounds, as new MMP-9 inhibitors,
are capable of protecting the endothelial integrity and decrease
the exacerbated vascular permeability, so may be advantageously
used to protect the endothelial barrier integrity in infections and
other diseases.
Example 5: Effect of Resv-LA (REV-LA) on TNF-.alpha. Release by
LPS-Activated Microglia
Cell Culture
BV-2 cells that are derived from raf/myc-immortalised murine
neonatal microglia are the most frequently used substitute for
primary microglia (Blasi et al., 1990).
BV-2 cells were maintained in 75 cm2 culture flasks in Dulbecco's
Modified Eagle's Medium (DMEM, Sigma) supplemented with 10% fetal
bovine serum (FBS, Sigma) and 1% Penicillin-Streptomycin solution
(Sigma), and cultured at 37.degree. C. in a humidified atmosphere
of 5% CO2.
Treatment of the BV-2 Microglial Cell Culture
BV-2 cells were plated on 24-well plates at a density of 105 cells
per well. On the following day cells were subjected to different
treatments. To study the effect of Resv-LA on TNF-alpha production
by LPS activated BV-2 cells, the cells were incubated in medium
containing 1 mg/ml LPS (Sigma-Aldrich) for 24 h, with or without
Resv-LA (REV-LA) 30 .mu.M.
TNF-Alpha Measurement
Supernatants from untreated and treated cells were centrifuged at
5000 rpm for 5 minutes and assayed for their TNF-.alpha. contents
using the TNF-.alpha. (mouse) AlphaLISA Detection Kit of
Perkin-Elmer. TNF-.alpha. released was normalized by cell number
and expressed as percentage of the maximal released obtained in the
LPS-stimulated condition.
Results
REV-LA (30 .mu.M) is efficient for reducing microglial activation
and neurotoxic TNF-.alpha. secretion by LPS-activated BV2 microglia
cells. Twenty-four hours after LPS treatment, TN-.alpha. expression
was reduced to 66.1.+-.4.2 in REV-LA-treated BV2.
Data are expressed as mean.+-.SEM. Statistical analysis of
differences between groups was performed by using unpaired
t-test/Mann-Whitney. The level of significance is set at p<0.05.
*P<0.05 and ***P<0.001 versus control (Ctr., non
LPS-challenged control), #P<0.05 ##P<0.001 versus
LPS-stimulated condition.
The results are presented in the FIG. 7. These data suggest that
Resv-LA (REV-LA) inhibits TNF-.alpha. release, which is a key
factor of inflammatory cascades as disclosed above, so Resv-LA may
advantageously be used for preventing or treating central nervous
system disorders.
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